The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of a DNA sequence of interest (such as DNA insert containing a natural or synthetic gene or gene fragment) into a pre-assembled cloning vector. The cloning vector includes all of the necessary components for replication of the DNA insert in a compatible host cell, e.g., promoter sequence, origin of replication sequence, termination sequence, and a selectable marker sequence. The DNA insert sequences can be derived from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA sequences. Alternatively, the DNA insert sequences can be created synthetically.
Insertion of the DNA sequence of interest can be accomplished by a number of techniques. The most common technique involves restriction enzymes. A restriction enzyme recognition site that is present in both the DNA insert and the vector of interest is cleaved with a restriction enzyme to provide for appropriate termini, the termini of either the DNA insert or the vector are treated with alkaline phosphatase to remove terminal phosphates and avoid undesirable joining, and the DNA sequence of interest is inserted into the vector at the compatible sites during a ligation reaction. A restriction enzyme site present in a pre-assembled vector must be compatible with a restriction enzyme site in the DNA sequence of interest.
Alternatively, the DNA of interest can be modified to obtain compatible restriction sites by filling in of cohesive ends as appropriate, or by the ligation of an appropriate oligonucleotide linker, which can be subsequently cleaved by the restriction enzyme of interest.
Conventional cloning methods can be time consuming and often involve multiple sub cloning steps. Therefore, a need exists for developing a simple and rapid method for synthesizing and identifying an optimal construct for use in a particular application.